Mäntsälä P, Zalkin H
J Biol Chem. 1979 Sep 10;254(17):8540-7.
Extracellular alpha-amylase was purified to homogeneity from a Marburg strain of Bacillus subtilis. The enzyme is a single polypeptide chain of molecular weight approximately 67,000. Its NH2-terminal amino acid sequence is Leu-Thr-Ala-Pro-Ser-Ile-Lys. A membrane-derived alpha-amylase was solubilizing from membrane vesicles by treatment with Triton X-100 and was highly purified by chromatography on an anti-alpha-amylase-protein A-Sepharose column. Membrane-derived alpha-amylase was indistinguishable from the soluble extracellular enzyme by sodium dodecyl sulfate-gel electrophoresis and radioimmunoassay. The membrane-derived enzyme contains phospholipid. Approximately 30 to 80% of the phospholipid was extracted from the purified enzyme by chloroform:methanol. The extracted phospholipid was predominately phosphatidylethanolamine. Treatment with phospholipase D released phosphatidic acid. Membrane-bound alpha-amylase was latent in membrane vesicles. Release of membrane-bound alpha-amylase from vesicles by an endogenous enzyme was maximal at pH 8.5, was inhibited by metal chelators and diisopropyl fluorophosphate and was stimulated by Ca2+ and Mg2+. The amount of membrane-bound alpha-amylase was related to the level of secretion.
从枯草芽孢杆菌马尔堡菌株中纯化得到了细胞外α-淀粉酶，使其达到了均一状态。该酶是一条分子量约为67,000的单多肽链。其氨基末端氨基酸序列为Leu-Thr-Ala-Pro-Ser-Ile-Lys。通过用Triton X-100处理，从膜泡中溶解出一种膜衍生的α-淀粉酶，并通过在抗α-淀粉酶-蛋白A-琼脂糖柱上进行色谱法进行了高度纯化。通过十二烷基硫酸钠-凝胶电泳和放射免疫测定，膜衍生的α-淀粉酶与可溶性细胞外酶无法区分。膜衍生的酶含有磷脂。通过氯仿：甲醇从纯化的酶中提取了约30%至80%的磷脂。提取的磷脂主要是磷脂酰乙醇胺。用磷脂酶D处理会释放出磷脂酸。膜结合的α-淀粉酶在膜泡中是潜伏性的。内源性酶从膜泡中释放膜结合的α-淀粉酶在pH 8.5时最大，受到金属螯合剂和二异丙基氟磷酸的抑制，并受到Ca2+和Mg2+的刺激。膜结合的α-淀粉酶的量与分泌水平有关。
